Solution preparation containing stabilized antibody

ABSTRACT

The present inventors discovered that a significant stabilization effect was achieved by using an acidic amino acid, aspartic acid or glutamic acid as a counter ion species in histidine buffer or tris(hydroxymethyl)aminomethane, specifically by using histidine-aspartate buffer or histidine-glutamate buffer, or tris(hydroxymethyl)aminomethane-aspartate or tris(hydroxy-methyl) aminomethane-glutamate as a buffer. The present inventors also discovered that a significant stabilization effect was achieved by using an acidic amino acid, aspartic acid or glutamic acid, as a counter ion species to a basic amino acid such as arginine, specifically by using arginine-aspartate or arginine-glutamate.

TECHNICAL FIELD

The present invention relates to antibody-containing formulations, inparticular to stable highly-concentrated antibody-containingformulations.

BACKGROUND ART

In recent years, there is an increasing demand for developingself-injectable antibody-containing formulations for subcutaneousinjection according to medical needs. Designing antibody-containingformulations for subcutaneous injection makes it necessary to increasethe antibody concentration in the administered solution, since a singledoses of antibody are very high (about 100 to 200 mg) and the injectionvolume for subcutaneous injection is generally limited.

Highly-concentrated antibody-containing solutions tend to form highlyviscous solutions by themselves due to intermolecular interactions andmacromolecular protein characteristics. Furthermore, degradationphenomenon such as aggregation becomes problematic when proteins arestored as highly-concentrated solutions, and thus, this degradation mustbe prevented. In particular, highly-concentrated antibody-containingsolutions tend to form aggregates during freeze-thawing, or when storedin liquid or frozen conditions for a long time (Non-patent Documents 1and 2).

To date, such highly-concentrated antibody-containing formulations aregenerally prepared by conventional lyophilizing concentration method(Patent Document 1), which is a method for stabilizinghighly-concentrated antibody-containing formulations. In the method,highly-concentrated antibody-containing formulations are obtained bylyophilizing an antibody solution of a relatively low concentration anddissolving in a smaller volume of water than the volume beforelyophilization. In this case, the increased viscosity of dissolvedformulations is of concern because a cryoprotectant such as sugar mustbe added to obtain lyophilized formulations.

In that aspect, this problem can be avoided when a liquid formulation isprepared without lyophilization. However, as described above,highly-concentrated antibody-containing liquid formulations tend to formaggregates. Nonetheless, such formulations are highly demanded becauseantibody-containing liquid formulations are easier to handle thanlyophilized formulations, and can be readily formulated into prefilledsyringe formulations.

There have been various studies to stabilize highly-concentratedantibody-containing liquid formulations (Non-patent Documents 1-4).Histidine buffer and arginine have been reported to be useful as abuffer and a stabilizer, respectively, in antibody-containing liquidformulations (Patent Documents 2, 3, 4, 5, and 6). The histidine bufferis commonly used in the form of hydrochloric acid salt. Recently, it hasbeen reported that histidine-acetate shows a higher stabilization effectthan histidine hydrochloride and thus acetic acid is useful as a counterion species in histidine buffer (Patent Document 6). Meanwhile, arginineas a stabilizer has been generally used in the form of argininehydrochloride. However, in some cases, sufficient stability is notobtained when hydrochloric acid or acetic acid is used as a counter ionspecies to histidine or arginine. Thus, more superior counter ionspecies are needed.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: WO 1997/004801-   Patent Document 2: WO 2008/121615-   Patent Document 3: WO 2009/141239-   Patent Document 4: WO 2008/071394-   Patent Document 5: WO 2006/065746-   Patent Document 6: WO 2006/044908

Non-Patent Documents

-   Non-patent Document 1: Challenges in the development of high protein    concentration formulations, J Pharm Sci, 2004, 93 (6), 1390-1402-   Non-patent Document 2: Curr Opin Biotechnol. 2009 December;    20(6):708-14. Epub 2009 Oct. 31-   Non-patent Document 3: Antibody structure, instability, and    formulation, J Pharm Sci, 2007, 96 (1), 1-26-   Non-patent Document 4: Formulation and delivery issues for    monoclonal antibody therapeutics, Adv Drug Del Rev, 2006, 58 (5-6),    686-706

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An objective of the present invention is to provide stablehighly-concentrated antibody-containing formulations which are suitablefor subcutaneous administration.

Means for Solving the Problems

To achieve the above-described objective, the present inventorsconducted dedicated studies. As a result, the present inventorsdiscovered that a significantly higher stabilization effect was achievedby using an acidic amino acid, aspartic acid or glutamic acid as acounter ion species in histidine buffer ortris(hydroxymethyl)aminomethane buffer, i.e., histidine-aspartate bufferor histidine-glutamate buffer, ortris(hydroxymethyl)aminomethane-aspartate buffer ortris(hydroxymethyl)aminomethane-glutamate buffer as compared toconventionally reported buffers for pharmaceutical formulations such ashistidine hydrochloride buffer and histidine-acetate buffer. The presentinventors also discovered that a significantly higher stabilizationeffect was achieved by using arginine-aspartate or arginine-glutamate asa stabilizer, i.e., by using an acidic amino acid, aspartic acid orglutamic acid as a counter ion species to a basic amino acid such asarginine which is used as a stabilizer as compared to conventionallyreported stabilizers for pharmaceutical formulations such as argininehydrochloride. Thus, the present inventors discovered that stablehighly-concentrated antibody-containing liquid formulations can beobtained by adding them as a stabilizer and thereby completed thepresent invention.

Specifically, the present invention provides:

-   -   [1] a stable antibody-comprising formulation comprising basic        amino acid-aspartate or basic amino acid-glutamate;    -   [2] the formulation of [1] comprising histidine-aspartate buffer        or histidine-glutamate buffer, wherein the basic amino acid is        histidine;    -   [3] the formulation of [1] comprising arginine-aspartate or        arginine-glutamate, wherein the basic amino acid is arginine;    -   [4] a stable antibody-comprising formulation comprising        histidine-aspartate buffer or histidine-glutamate buffer, and        arginine-aspartate or arginine-glutamate;    -   [5] a stable antibody-comprising formulation comprising        tris(hydroxymethyl)aminomethane-aspartate buffer or        tris(hydroxymethyl)aminomethane-glutamate buffer;    -   [6] a stable antibody-comprising formulation comprising        tris(hydroxymethyl)aminomethane-aspartate buffer and        tris(hydroxymethyl)aminomethane-glutamate buffer;    -   [7] the formulation according to any one of [1] to [6], which        does not substantially comprise chloride ion and acetate ion;    -   [8] the formulation according to any one of [1] to [7], which        additionally comprises a sugar;    -   [9] the formulation according to any one of [1] to [8], wherein        the antibody is a humanized antibody or a human antibody;    -   [10] the formulation according to any one of [1] to [9], wherein        the antibody has been modified to have an isoelectric point (pI)        of 5 to 8;    -   [11] the formulation according to any one of [1] to [10],        wherein the antibody concentration is 50 mg/ml or more;    -   [12] the formulation according to any one of [1] to [10],        wherein the antibody concentration is 50 to 250 mg/ml;    -   [13] the formulation according to any one of [1] to [12], which        is a liquid formulation;    -   [14] the formulation according to [13], wherein the viscosity of        the liquid formulation is 30 mPa s or less;    -   [15] the formulation according to [13] or [14], wherein the        liquid formulation is stable at 2° C. to 8° C. for at least six        months;    -   [16] the formulation according to any one of [13] to [15], which        has not been subjected to lyophilization during preparation of        the formulation;    -   [17] the formulation according to any one of [13] to [16], which        is frozen stored at −30° C. to −10° C.;    -   [18] the formulation according to any one of [1] to [12],        wherein the formulation is a lyophilized formulation;    -   [19] the formulation according to any one of [2], [4], and [7]        to [18], wherein the buffer concentration is 5 to 100 mM;    -   [20] the formulation according to any one of [3] and [7] to        [19], wherein the arginine concentration is 5 to 300 mM;    -   [21] the formulation according to any one of [1] to [20],        wherein the antibody is an anti-IL-6 receptor antibody;    -   [22] the formulation according to any one of [2], [4], and [7]        to [21], wherein the buffer substantially comprises only amino        acid(s);    -   [23] the formulation according to any one of [1] to [22], which        is for subcutaneous administration;    -   [24] a method for suppressing aggregation formation during        frozen storage of a highly-concentrated antibody-comprising        formulation by using aspartic acid or glutamic acid as a counter        ion species to a buffer in the formulation;    -   [25] a method for suppressing aggregation formation during        liquid storage of a highly-concentrated antibody-comprising        formulation by using aspartic acid or glutamic acid as a counter        ion species to a buffer in the formulation;    -   [26] a method for suppressing aggregation formation during        frozen storage of a highly-concentrated antibody-comprising        formulation by using aspartic acid or glutamic acid as a counter        ion species to a stabilizer in the formulation; and    -   [27] a method for suppressing aggregation formation during        liquid storage of a highly-concentrated antibody-comprising        formulation by using aspartic acid or glutamic acid as a counter        ion species to a stabilizer in the formulation.

Furthermore, the present invention relates to use of basic aminoacid-aspartate or basic amino acid-glutamate in manufacturing a stableantibody-comprising formulation; and aspartic acid or glutamic acid as acounter ion species to a buffer or stabilizer in a highly-concentratedantibody-comprising formulation, used in a method for suppressingaggregation during frozen storage or liquid storage of the formulation.

Effects of the Invention

The present invention provides antibody-containing formulations that aresuperior in stability. The present invention can also providehighly-concentrated antibody-containing formulations by suppressing theaggregation formation in liquid and frozen formulations. Thehighly-concentrated antibody-containing formulations of the presentinvention can be stably stored in liquid or in frozen condition for along period. Furthermore, the formulations of the present invention havean improved stability against freeze-thawing stress. In addition, interms of osmotic pressure, stabilization can be achieved withoutincreasing osmotic pressure, by using aspartic acid and glutamic acidrather than the conventionally used hydrochloric acid and acetic acid ascounter ion species to histidine, arginine, ortris(hydroxymethyl)aminomethane. It is advantageous to achievestabilization without increasing the osmotic pressure, when one intendsto produce almost isotonic, stable formulations, such as formulationsfor subcutaneous (SC) administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab1 at 40° C. on the vertical axis.

FIG. 2 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab2 at 40° C. on the vertical axis.

FIG. 3 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing of Mab1 on the vertical axis.

FIG. 4 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab1 at 25° C. on the vertical axis.

FIG. 5 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab1 at 5° C. on the vertical axis.

FIG. 6 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing (−20° C. to room temperature) of Mab1on the vertical axis.

FIG. 7 is a graph plotted with the amount (%) of aggregate after threemonths of storage of Mab1 at −20° C. on the vertical axis.

FIG. 8 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing (−20° C. to room temperature) of Mab2on the vertical axis.

FIG. 9 is a graph plotted with the amount (%) of aggregate after storageof Mab1 at −20° C. on the vertical axis.

FIG. 10 is a graph plotted with the amount (%) of aggregate afterfreeze-thawing (−20° C. to room temperature) of Mab1 on the verticalaxis.

FIG. 11 is a graph plotted with the amount (%) of aggregate after threemonths of storage of Mab1 at 25° C. on the vertical axis.

FIG. 12 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab2 at 25° C. on the vertical axis.

FIG. 13 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab3 at 25° C. on the vertical axis.

FIG. 14 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing (between −20° C. and roomtemperature) of Mab3 on the vertical axis.

FIG. 15 is a graph plotted with time dependent changes of the amount (%)of aggregate during storage of Mab4 at 25° C. on the vertical axis.

FIG. 16 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing (between −20° C. and roomtemperature) of Mab4 on the vertical axis.

FIG. 17 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab1, Mab2, and Mab3 at 25° C. on thevertical axis.

FIG. 18 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing (between −20° C. and roomtemperature) of Mab1, Mab2, and Mab3 on the vertical axis.

FIG. 19 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab5 at 25° C. on the vertical axis.

FIG. 20 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing (between −20° C. and roomtemperature) of Mab5 on the vertical axis.

FIG. 21 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab1, Mab2, Mab3, Mab4, and Mab5 at 25°C. on the vertical axis.

FIG. 22 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing (between −20° C. and roomtemperature) of Mab1, Mab2, Mab3, Mab4, and Mab5 on the vertical axis.

FIG. 23 is a graph plotted with time-dependent changes of the amount (%)of aggregate during storage of Mab1, Mab2, and Mab3 at 25° C. on thevertical axis.

FIG. 24 is a graph plotted with time-dependent changes of the amount (%)of aggregate during freeze-thawing (between −20° C. and roomtemperature) of Mab1, Mab2, and Mab3 on the vertical axis.

MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described more specifically.

The present invention provides stable antibody-containing formulationswhich contain basic amino acid-aspartate or basic amino acid-glutamate.In the present invention, basic amino acids include, for example,histidine, arginine, and lysine. Furthermore, in the present invention,buffers of basic amino compounds such as tris(hydroxymethyl)aminomethaneare also included in the definition of the basic amino acids of thepresent invention.

Specifically, the present invention provides stable antibody-containingformulations which contain histidine-aspartate buffer orhistidine-glutamate buffer where the basic amino acid is histidine.Furthermore, the present invention provides stable antibody-containingformulations which contain arginine-aspartate or arginine-glutamate as astabilizer, where the basic amino acid is arginine. The presentinvention also provides stable antibody-containing formulations whichcontain histidine-aspartate buffer or histidine-glutamate buffer, andarginine-aspartate or arginine-glutamate. Furthermore, the presentinvention provides stable antibody-containing formulations which containtris(hydroxymethyl)aminomethane-aspartate buffer ortris(hydroxymethyl)aminomethane-glutamate buffer. The present inventionalso provides stable antibody-containing formulations which containtris(hydroxymethyl)aminomethane-aspartate buffer andtris(hydroxymethyl)aminomethane-glutamate buffer. Theantibody-containing formulation of the present invention refers to aformulation that contains an antibody as an active ingredient and isprepared in a form that allows administration to animals such as human.

Herein, “stable antibody-containing formulation” refers to a formulationin which aggregation of proteins such as antibody is hardly formed,specifically, a formulation in which degradation such as formation ofinsoluble and soluble aggregates hardly occurs during storage in liquidor in frozen condition.

The concentration of antibody in a formulation of the present inventionis not particularly limited; however, the formulation preferablycontains a high concentrated antibody. The antibody concentration ispreferably 50 mg/ml or more, more preferably 100 mg/ml or more, evenmore preferably 120 mg/ml or more, still more preferably 150 mg/ml ormore, and yet more preferably 180 mg/ml or more. The upper limit of theantibody concentration in a formulation of the present invention is notparticularly limited; however, the limit is generally 250 mg/ml.

The antibodies used in the present invention are not particularlylimited, as long as they bind to an antigen of interest. The antibodiesmay be polyclonal or monoclonal antibodies; however, monoclonalantibodies are preferred because they can be stably produced ashomogeneous antibodies.

The monoclonal antibodies used in the present invention include not onlythose derived from animals such as humans, mice, rats, hamsters,rabbits, sheep, camels, and monkeys, but also artificially modified generecombinant antibodies such as chimeric antibodies, humanizedantibodies, and bispecific antibodies. The antibodies of the presentinvention also include gene recombinant antibodies that result fromartificially modifying the antibody constant regions to alter thephysical properties of the antibody molecule (specifically, alterationof the isoelectric point (pI), improvement of the affinity for Fcreceptor, etc) for the purpose of improving the blood persistence and invivo pharmacokinetics.

The immunoglobulin class of the antibodies used in the present inventionis not particularly limited; and the class may be any class, includingIgG such as IgG1, IgG2, IgG3, and IgG4, IgA, IgD, IgE, and IgM. However,IgG and IgM are preferred.

The antibodies used in the present invention also include not only wholeantibodies but also antibody fragments such as Fv, Fab, and F(ab)2, andminibodies (low molecular weight antibodies) such as monovalent orbivalent single-chain Fv that result from linking antibody variableregions via a linker such as peptide linker (scFv, sc(Fv)2, diabodiessuch as scFv dimer, etc).

The above-described antibodies used in the present invention can beprepared by methods known to those skilled in the art.

Basically, monoclonal antibody-producing hybridomas can be prepared bythe conventional methods described below. Specifically, immunization iscarried out by a conventional immunization method using a desiredantigen or cells expressing the desired antigen as a sensitizingantigen. The prepared immunocytes are fused with known parental cells bya conventional cell fusion method. The fused cells are screened formonoclonal antibody-producing cells (hybridomas) by conventionalscreening methods. Hybridomas can be generated, for example, accordingto the method of Milstein et al. (Kohler, G. and Milstein, C., MethodsEnzymol. (1981) 73:3-46). When an antigen has low immunogenicity,immunization can be performed using the antigen linked to immunogenicmacromolecules such as albumin.

Alternatively, it is possible to use gene recombinant antibodiesproduced using gene recombination techniques in which antibody genes arecloned from hybridomas and inserted into appropriate vectors, and theresulting vectors are introduced into hosts (see, for example, Carl, A.K. Borrebaeck, James, W. Larrick, Therapeutic Monoclonal Antibodies,Published in the United Kingdom by Macmillan Publishers, 1990).Specifically, cDNAs for antibody variable regions (V regions) aresynthesized from the mRNAs of hybridomas using reverse transcriptase.When a DNA encoding an antibody V region of interest is obtained, theDNA is linked to a DNA encoding a desired antibody constant region (Cregion). The resulting construct is inserted into an expression vector.Alternatively, the antibody V region-encoding DNA may be inserted intoan expression vector carrying the DNA of the antibody C region. Theresulting construct is inserted into an expression vector so that it isexpressed under the control of an expression regulatory region, forexample, enhancer and promoter. Then, host cells are transformed withthe expression vector to express the antibody.

In the present invention, artificially modified gene recombinantantibodies such as chimeric and humanized antibodies can be used toreduce heterologous antigenicity against human. Such modified antibodiescan be produced using known methods. A chimeric antibody is an antibodyhaving the heavy-chain and light-chain variable regions of an antibodyfrom a nonhuman mammal such as mouse, and the heavy-chain andlight-chain constant regions of a human antibody. The chimeric antibodycan be produced by linking a DNA encoding the variable regions of amouse antibody to a DNA encoding the constant regions of a humanantibody, inserting the ligate into an expression vector, and thenintroducing the vector into a host for expression.

A humanized antibody is also referred to as reshaped human antibody, andis obtained by substituting the complementarity determining region (CDR)of a human antibody for the complementarity determining region of anantibody derived from a nonhuman mammal, for example, mouse.Conventional gene recombination techniques are known. Specifically, aDNA sequence is designed to have a mouse antibody CDR linked to a humanantibody framework (FR) region, and is synthesized by PCR using severaloligonucleotides prepared to have overlapping regions at their ends. Theobtained DNA is ligated to a DNA encoding a human antibody constantregion and then inserted into an expression vector. The expressionvector is introduced into a host to produce the humanized antibody (see,European Patent Application Publication No. EP 239400 and WO 96/02576).The CDR-linked human antibody FR is selected so that the complementaritydetermining region forms a preferable antigen-binding domain. Aminoacids in the framework region of the antibody variable region can besubstituted as required so that the complementarity determining regionof the reshaped human antibody forms a suitable antigen-binding domain(Sato, K. et al., Cancer Res. (1993) 53, 851-856).

There are known techniques for substituting amino acids in antibodies toimprove antibody activities, physical properties, pharmacokinetics,safety, and such. Examples of such techniques are described below. Theantibodies of the present invention also include those having such aminoacid substitutions.

Techniques are reported for substituting amino acids in the IgG antibodyvariable regions, and include humanization (Tsurushita N, Hinton P R,Kumar S., Design of humanized antibodies: from anti-Tac to Zenapax.,Methods. 2005 May; 36(1):69-83); affinity maturation to enhance thebinding activity via amino acid substitution in the complementaritydetermining region (CDR) (Rajpal A, Beyaz N, Haber L, Cappuccilli G, YeeH, Bhatt R R, Takeuchi T, Lerner R A, Crea R., A general method forgreatly improving the affinity of antibodies by using combinatoriallibraries., Proc Natl Acad Sci USA. 2005 Jun. 14; 102(24):8466-71); andimprovement of physicochemical stability via amino acid substitution inthe framework (FR) (Ewert S, Honegger A, Pluckthun A., Stabilityimprovement of antibodies for extracellular and intracellularapplications: CDR grafting to stable frameworks and structure-basedframework engineering., Methods. 2004 October; 34(2):184-99. Review).There are also known techniques for enhancing antibody-dependent cellcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) bysubstituting amino acids in IgG antibody Fc domain (Kim S J, Park Y,Hong H J., Antibody engineering for the development of therapeuticantibodies., Mol Cells. 2005 Aug. 31; 20(1):17-29. Review). Furthermore,in addition to techniques for enhancing the effector functions, thereare reports published on techniques for improving the antibody half-lifein blood by substituting amino acids in Fc (Hinton P R, Xiong J M,Johlfs M G, Tang M T, Keller S, Tsurushita N., An engineered human IgG1antibody with longer scrum half-life., J Immunol. 2006 Jan. 1;176(1):346-56; Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, MedesanC, Ober R J, Ward E S., Increasing the serum persistence of an IgGfragment by random mutagenesis, Nat Biotechnol. 1997 July;15(7):637-40). Another known technique includes amino acid substitutiontechnique to control the isoelectric point (pI) of an antibody for thepurpose of improving the blood persistence or in vivo pharmacokinetics,specifically, a technique for modifying amino acid residues exposed onthe surface of an antibody to control the pI of the antibody (WO07/114319). Various techniques to substitute amino acids in the constantregions for the purpose of improving the physical properties of anantibody are also known (WO 09/41613).

Reduction of the dosage of antibody as a pharmaceutical or extension ofthe interval of antibody administration can be expected by extending thehalf-life or plasma retention of an antibody. Promising technologies toachieve this include a technique for decreasing the isoelectric point ofantibody (WO 07/114319). The formulations of the present invention havea high stabilizing effect for antibodies with an altered isoelectricpoint. The pI-modified antibody refers to a modified antibody whose pIis lower than that of the original antibody by one or more, preferablytwo or more, and more preferably three or more. In general, natural (orordinary) antibodies are assumed to have an isoelectric point within therange of 7.5 to 9.5. The formulations of the present invention have ahigh stabilizing effect for, in particular, antibodies with a lowisoelectric point which hardly exist in nature. The isoelectric point ofsuch antibodies may be 5.0 to 8.0, preferably 5.0 to 7.5, morepreferably 5.0 to 7.0, and still more preferably 5.5 to 6.5. Asdescribed in the Examples below, the isoelectric point of Mab1, whichwas produced from modification of the Mab2 (isoelectric point=9.3) aminoacid sequence to control the isoelectric point, was 5.8.

Methods for obtaining human antibodies are also known. For example,desired human antibodies with antigen-binding activity can be obtainedby sensitizing human lymphocytes with an antigen of interest or cellsexpressing an antigen of interest in vitro; and fusing the sensitizedlymphocytes with human myeloma cells such as U266 (see Japanese PatentApplication Kokoku Publication No. (JP-B) H01-59878 (examined, approvedJapanese patent application published for opposition)). Alternatively,desired human antibodies can also be obtained by immunizing transgenicanimals having the entire repertoire of human antibody genes with anantigen (see WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO96/34096, and WO 96/33735). Furthermore, techniques for obtaining humanantibodies by panning with a human antibody library are known. Forexample, the variable regions of human antibodies can be expressed assingle-chain antibodies (scFvs) on the surface of phages using a phagedisplay method, and then phages that bind to the antigen can beselected. The genes of selected phages can be analyzed to determine theDNA sequences that encode the variable regions of human antibodies thatbind to the antigen. When the DNA sequences of scFvs that bind to theantigen are identified, appropriate expression vectors carrying thesesequences can be constructed to obtain human antibodies. Such methodsare already well known. See WO 92/01047, WO 92/20791, WO 93/06213, WO93/11236, WO 93/19172, WO 95/01438, and WO 95/15388. The antibodies usedin the present invention also include such human antibodies.

When the antibody genes are isolated and introduced into appropriatehosts to produce antibodies, hosts and expression vectors can be used inappropriate combinations. When eukaryotic cells are used as a host,animal cells, plant cells, and fungal cells can be used. The animalcells include: (1) mammalian cells such as CHO, COS, myeloma, babyhamster kidney (BHK), HeLa, and Vero cells; (2) amphibian cells such asXenopus oocytes; and (3) insect cells such as sf9, sf21, and Tn5. Knownplant cells include cells derived from genus Nicotiana such as Nicotianatabacum, which can be cultured as a callus. Known fungal cells includeyeasts such as genus Saccharomyces, for example Saccharomycescerevisiae, and filamentous fungi such as genus Aspergillus, for exampleAspergillus niger. When using prokaryotic cells, production systemsusing bacterial cells can be used. Known bacterial cells includeEscherichia coli (E. coli) and Bacillus subtilis. The antibodies can beobtained by introducing the antibody genes of interest into these cellsby transformation and then culturing the transformed cells in vitro.

The antibodies used in the present invention also include antibodyfragments, minibodies, and modified antibodies. Such antibody fragmentsand minibodies include, for example, Fab, F(ab′)2, Fv, or mono-, bi-, ormulti-valent single-chain Fv (scFv, sc(Fv)2, or such) resulting fromlinking H chain and L chain Fvs via appropriate linkers (Huston J. S. etal., (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883). Specifically,such antibody fragments are generated by treating antibodies with anenzyme such as papain or pepsin. Alternatively, the gene encoding anantibody fragment is constructed, inserted into an expression vector,and expressed in appropriate host cells (see, for example, Co, M. S. etal., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H.,Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A.,Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol.(1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121,663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9,132-137).

Modified antibodies include antibodies linked to polyethylene glycol(PEG) or various molecules such as cytotoxic agents (Farmaco. 1999 Aug.30; 54(8):497-516; Cancer J. 2008 May-June; 14(3):154-69). The“antibodies” of the present invention also include such modifiedantibodies. Such modified antibodies can be prepared by chemicallymodifying the obtained antibodies. Such methods are already establishedin this field.

Antibodies to be contained in formulations of the present inventioninclude, but are not limited to, anti-tissue factor antibodies,anti-IL-6 receptor antibodies, anti-IL-6 antibodies, anti-HM1.24 antigenmonoclonal antibodies, anti-parathyroid hormone-related peptideantibodies (anti-PTHrP antibodies), anti-glypican-3 antibodies,anti-ganglioside GM3 antibodies, anti-TPO receptor agonist antibodies,antibodies as a functional substitute for coagulation factor VIII,anti-IL31 receptor antibodies, anti-HLA antibodies, anti-AXL antibodies,anti-CXCR4 antibodies, anti-NR10 antibodies, and bispecific antibodiesagainst factor IX and factor X.

Preferred reshaped humanized antibodies used in the present inventioninclude humanized interleukin 6 (IL-6) receptor antibodies (tocilizumab,hPM-1, and MRA) (see WO 92/19759), humanized anti-HM1.24 antigenmonoclonal antibodies (see WO 98/14580), humanized anti-parathyroidhormone-related peptide antibodies (anti-PTHrP antibodies) (see WO98/13388), humanized anti-tissue factor antibodies (see WO 99/51743),humanized anti-glypican-3 IgGiK antibodies (see PCT/JP05/013103), andanti-NR10 humanized antibodies (see WO2009/072604). Particularlypreferred humanized antibodies used in the present invention arehumanized anti-IL-6 receptor antibodies.

Preferred human IgM antibodies include recombinant humananti-ganglioside GM3 IgM antibodies (see WO 05/05636).

Preferred minibodies include anti-TPO receptor agonist diabodies (see WO02/33072) and anti-CD47 agonist diabodies (see WO 01/66737).

Furthermore, antibodies with an improved isoelectric point include, forexample, Mab1 (H chain/SEQ ID NO: 1; L chain/SEQ ID NO: 2), which is ananti-IL-6 receptor antibody described in WO 2009/041621, anti-NR10humanized antibodies, and fully humanized NS22 antibodies produced bythe method described in Example 12 of WO2009/072604.

In a preferred embodiment, the buffer of a formulation of the presentinvention (for example, histidine-aspartate buffer, histidine-glutamatebuffer, tris(hydroxymethyl)aminomethane-aspartate buffer, ortris(hydroxymethyl)aminomethane-glutamate buffer) is prepared bytitrating an aqueous solution containing basic amino acid such ashistidine or tris(hydroxymethyl)aminomethane as a free amino acid withan aqueous solution containing aspartic acid and/or glutamic acid as afree amino acid. Alternatively, the buffer can be prepared by adding theingredients in the reverse order, or by direct titration with powders.

In a preferred embodiment, arginine-aspartate or arginine-glutamate in aformulation of the present invention is a salt prepared by titrating anaqueous solution containing aspartic acid (free amino acid) and/orglutamic acid (free amino acid) as a free amino acid with an aqueoussolution containing arginine (free base) as a free amino acid.Alternatively, the salt can be prepared by adding the ingredients in thereverse order, or by direct titration with powders.

The present inventors conducted freeze-thawing study, thermalacceleration study, long term stability study, and frozen storage studyto assess the effects of various additives on the stability ofhighly-concentrated antibody formulations during storage. As a result,the present inventors discovered that the aggregation formation wassignificantly suppressed by using an acidic amino acid, aspartic acid orglutamic acid as a counter ion species in histidine buffer, i.e., byusing histidine-aspartate buffer or histidine-glutamate buffer as abuffer as compared to conventional buffers for pharmaceuticalformulations such as histidine hydrochloride buffer andhistidine-acetate buffer.

The present inventors also discovered that a higher stabilization effectwas achieved by adding arginine-aspartate or arginine-glutamate ascompared to arginine hydrochloride which has been reported as astabilizer for antibody-containing formulations. The assessment resultsare exemplified in the Examples hereinbelow, using a humanized anti-IL-6receptor antibody.

Specifically, stable highly-concentrated antibody-containingformulations that have low levels of antibody aggregation can beprepared by adding histidine-aspartate buffer or histidine-glutamatebuffer, or tris(hydroxymethyl)aminomethane-aspartate buffer ortris(hydroxymethyl)aminomethane-glutamate buffer. Furthermore, morestable highly-concentrated antibody-containing formulations that havemuch lower levels of antibody aggregation can be prepared by addingarginine-aspartate or arginine-glutamate as a stabilizer. Thus, thepresent invention relates to methods for significantly suppressing theaggregation formation by using histidine-aspartate buffer orhistidine-glutamate buffer, or tris(hydroxymethyl)aminomethane-aspartatebuffer or tris(hydroxymethyl)aminomethane-glutamate buffer as a bufferfor highly-concentrated antibody-containing solutions, and methods forsignificantly suppressing the aggregation formation by addingarginine-aspartate or arginine-glutamate as a stabilizer tohighly-concentrated antibody-containing solutions.

In an embodiment, the methods of the present invention include, forexample, methods for suppressing the aggregation formation duringstorage of highly-concentrated antibody-containing formulations underfrozen conditions or freeze-thawing by using aspartic acid or glutamicacid as a counter ion species to the buffer (for example, histidinebuffer or tris(hydroxymethyl)aminomethane buffer) or as a counter ionspecies to the stabilizer (for example, arginine) in the formulations.

In another embodiment, the methods of the present invention include, forexample, methods for suppressing the aggregation formation duringstorage of highly-concentrated antibody-containing formulations in aliquid condition by using aspartic acid or glutamic acid as a counterion species to the buffer (for example, histidine buffer ortris(hydroxymethyl)aminomethane buffer) or as a counter ion species tothe stabilizer (for example, arginine) in the formulations.

In the present invention, buffers that can be used instead of histidinebuffer and in which aspartic acid or glutamic acid is used as a counterion species include tris(hydroxymethyl)aminomethane (Tris) andimidazole. Such buffers can also be added to the histidine buffer of thepresent invention.

In the present invention, stabilizers to which aspartic acid or glutamicacid can be used as a counter ion species include argininamide, lysine,meglumine, spermine, spermidine, magnesium, calcium, sodium, andpotassium, in addition to arginine.

As described above, the present invention provides stableantibody-containing formulations that comprise histidine-aspartatebuffer or histidine-glutamate buffer, ortris(hydroxymethyl)aminomethane-aspartate buffer ortris(hydroxymethyl)aminomethane-glutamate buffer. A much higherstabilization effect is achieved in the antibody-containing formulationsof the present invention when arginine-aspartate or arginine-glutamateis additionally contained. Thus, the present invention relates toantibody-containing formulations which contain a salt that combines abasic amino acid such as histidine or arginine (preferably, histidineand/or arginine) or tris(hydroxymethyl)aminomethane with aspartic acidor glutamic acid in liquid.

The histidine used in the present invention may be histidine itself or aderivative thereof, and L-histidine is particularly preferred. Thearginine used in the present invention may be arginine itself, aderivative thereof, or a salt thereof, and L-arginine or a salt thereofis particularly preferable. Preferred salts of arginine includeaspartate salt and glutamate salt.

In the formulations of the present invention, the concentration (amount)of histidine-aspartate buffer or histidine-glutamate buffer ispreferably 1 to 100 mM, more preferably 5 to 100 mM, even morepreferably 5 to 50 mM, and still more preferably 10 to 25 mM.

In the formulations of the present invention, the concentration (amount)of arginine is preferably 5 to 300 mM, more preferably 25 to 200 mM, andstill more preferably 50 to 150 mM.

The formulations of the present invention may be solutions(antibody-containing liquid formulations) or lyophilized formulations.The liquid formulations of the present invention also include solutionsbefore lyophilization step(s), and the dissolved solutions. The liquidformulations of the present invention are preferably produced withoutthe lyophilization step(s) in production. Meanwhile, the lyophilizedformulations of the present invention can be obtained by lyophilizingthe liquid formulations of the present invention using methods known tothose skilled in the art.

The pH of the formulations of the present invention is preferably 4 to8, more preferably 5.0 to 7.5, and still more preferably 5.5 to 6.5.

The viscosity of the liquid formulations of the present invention atroom temperature (25° C.) is preferably 30 mPa s or less, morepreferably 20 mPa s or less, and still more preferably 15 mPa s or less.

Significant changes are not observed for the liquid formulations of thepresent invention for at least 6 months, preferably 12 months, morepreferably two years, even more preferably three years at refrigerationtemperature (2° C. to 8° C.), or for at least six months, preferably oneyear, and more preferably two years at room temperature (22° C. to 28°C.). Specifically, the present invention relates to liquid formulationsthat are stable for at least six months at 22° C. to 28° C.

The liquid formulations of the present invention can be frozen andstored at a temperature within the range of ˜30° C. to −10° C.

The formulations of the present invention may additionally containsurfactants. As used in the present invention, preferred surfactantsinclude polyoxyethylene sorbitan fatty acid esters and polyoxyethylenepolyoxypropylene alkyl ethers, and more preferred surfactants includepolysorbates 20 and 80, and Pluronic F-68 (poloxamer 188). Thesurfactants can be added to the formulations of the present invention ingeneral at 0.0001% to 10% (w/v), preferably at 0.001% to 5%, and morepreferably at 0.005% to 3%.

The formulations of the present invention may further contain aminoacids. As used in the preset invention, preferred amino acids includenatural amino acids and amino acid derivatives, and particularlypreferred amino acids include L-methionine and L-proline.

The formulations of the present invention may further contain sugars.The preferred sugars used in the present invention include sucrose,trehalose, meglumine, and sorbitol.

The amount of amino acid or sugar that can be added to the formulationsof the present invention is generally 1 to 1000 mM, preferably 5 to 500mM, and more preferably 10 to 300 mM.

The formulations of the present invention may further contain inorganicsalts. The preferred inorganic salts used in the present inventioninclude magnesium salts and calcium salts.

The formulations of the present invention are substantially constitutedby the ingredients of A to D below.

-   -   (A) anti-IL-6 receptor antibody;    -   (B) histidine-aspartate buffer and/or histidine-glutamate        buffer;    -   (C) arginine (including arginine-aspartate and        arginine-glutamate), amino acids other than arginine, and/or        sugars as needed; and    -   (D) surfactants.

“Substantially constituted” means that the concentrations of theoptional additives described below, which are ingredients other than theingredients generally added to the formulations, are 5 mM or less,preferably 2 mM or less, and more preferably 1 mM or less. Such optionaladditives include cryoprotectants, suspending agents, solubilizingagents, isotonizing agents, preservatives, adsorption inhibitors,diluents, excipients, pH adjustors, analgesics, sulfur-containingreducing agents, and antioxidants.

Furthermore, it is preferred that the formulations of the presentinvention do not contain anions other than aspartic acid and glutamicacid, as counter ions to the buffer or stabilizer. In an embodiment,such formulations include, for example, those that do not substantiallycontain chloride ion and acetate ion. “Substantially do not containchloride ion and acetate ion” means that the concentrations of chlorideion and acetate ion are, for example, 5 mM or less, preferably 2 mM orless, and more preferably 1 mM or less. Highly stableantibody-containing formulations can be produced without increasing theosmotic pressure, as a result of using aspartic acid and glutamic acidwhich have higher stabilizing effect as a counter ions and notsubstantially including chloride ion and acetate ion with poorerstabilization effect.

If needed, the formulations of the present invention may further containappropriate cryoprotectants, suspending agents, solubilizing agents,isotonizing agents, preservatives, adsorption inhibitors, diluents,excipients, pH adjustors, analgesics, sulfur-containing reducing agents,antioxidants, and such.

Cryoprotectants include, for example, sugars such as trehalose, sucrose,and sorbitol.

Solubilizing agents include, for example, polyoxyethylene hydrogenatedcastor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitanmonolaurate, macrogol, and castor oil fatty acid ethyl ester.

Isotonizing agents include, for example, sodium chloride, potassiumchloride, and calcium chloride.

Preservatives include, for example, methyl parahydroxybenzoate, ethylparahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

Adsorption inhibitors include, for example, human serum albumin,lecithin, dextran, ethylene oxide/propylene oxide copolymer,hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenatedcastor oil, and polyethylene glycol.

Sulfur-containing reducing agents include, for example, those containingsulfhydryl groups such as N-acetylcysteine, N-acetylhomocysteine,thioctic acid, thiodiglycol, thioethanol amine, thioglycerol,thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate,glutathione, and thioalkanoic acids having one to seven carbon atoms.

Antioxidants include, for example, erythorbic acid,dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherolacetate, L-ascorbic acid and salts thereof, L-ascorbic acid palmitate,L-ascorbic acid stearate, sodium hydrogen sulfite, sodium sulfite,triamyl gallate, propyl gallate, and chelating agents such as disodiumethylenediamine tetraacetate (EDTA), sodium pyrophosphate, and sodiummetaphosphate.

The formulations of the present invention may be administered orally orparenterally. In general, the formulations are administeredparenterally, specifically via injection, transdermal administration,transmucosal administration, nasal administration, pulmonaryadministration, or the like. Injection includes, for example, systemicand local administrations by subcutaneous injection, intravenousinjection, intramuscular injection, or such. The injection volume islimited in subcutaneous injection; however, a single antibody dose maybe a large quantity (about 100 to 200 mg). Thus, the formulations of thepresent invention are particularly suitable for subcutaneousadministration (injection).

In terms of pain, it is preferred that the osmotic pressure ratio ofbuffering agent is close to isotonic 1.0 in formulations forsubcutaneous administration. Thus, the osmotic pressure ratio of theliquid formulations of the present invention is preferably about 1.Arginine, sugars, and such are added to improve the stability offormulations during storage. However, when the osmotic pressure isgreater than the isotonic level, it may cause pain of subcutaneousadministration. Thus, adding such stabilizers with consideration ofosmotic pressure is preferred.

Furthermore, the present invention relates to use of basic aminoacid-aspartate or basic amino acid-glutamate in manufacturing a stableantibody-comprising formulation; and aspartic acid or glutamic acid as acounter ion species to a buffer or stabilizer in a highly-concentratedantibody-comprising formulation, used in a method for suppressingaggregation during frozen storage or liquid storage of the formulation.

All prior-art documents cited in the specification are incorporatedherein by reference.

EXAMPLES

Hereinbelow, the present invention is specifically described withreference to the Examples, but the scope of the present invention is notto be construed as being limited thereto.

Example 1 Assessment of the Stabilization Effect of Counter Ion SpeciesUsing Mab1 and Mab2

Mab1 (H chain/SEQ ID NO: 1; L chain/SEQ ID NO: 2) and Mab2 (H chain/SEQID NO: 3; L chain/SEQ ID NO: 4; tocilizumab), which are described as ananti-IL-6 receptor antibody in WO 2009/041621, were expressed by amethod known to those skilled in the art using a stable expression CHOcell line, and then purified to high purity by a method known to thoseskilled in the art using protein A. Purified Mab1 and Mab2 were used inthe stability study described in the Examples below.

The stability of two types of formulations, containinghistidine-chloride or histidine-acetate, was assessed by freeze-thawingor by storage at 40° C. using Mab1 and Mab2. Mab1 and Mab2 formulationswere prepared by overnight dialysis against each formulated solution(Table 1), followed by concentration of the solutions. The finalconcentrations of Mab1 and Mab2 were adjusted to 37 mg/ml. Thefreeze-thawing study was carried out by conducting ten cycles of slowfreeze-thawing (freezing at −20° C. followed by thawing at roomtemperature), and then ten cycles of rapid freeze-thawing (freezing at−20° C. followed by thawing in warm water bath (37° C.)). The amount ofaggregate in each formulation after freeze-thawing or storage at 40° C.was calculated by the area percentage method using size exclusionchromatography (SEC). An increase of the aggregates (%) suggests reducedstability of Mab1 or Mab2. Thus, increase in the amount of aggregate wasused as an indicator to compare the stability between respectiveformulations.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography (SEC) was performed to analyze thequantity of aggregates and low-molecular-weight degradation products ineach formulation. Each formulation was diluted to about 0.4-2.0 mg/mlwith the mobile phase described below, and then analyzed usingG3000SWXI, column (Tosoh Co.) at a flow rate of 0.5 ml/min with a mobilephase of 50 mM phosphate buffer (pH 7.0) containing 300 mM NaCl(detection wavelength: 220 nm). The elution peak that appeared earlierthan the monomer peak was analyzed as the aggregates, and the elutionpeak that appeared after the monomer peak but earlier than thebuffer-derived peak was analyzed as the low-molecular-weight degradationproducts. The respective contents (%) were calculated by the areapercentage method.

TABLE 1 Formulation list No. Buffer Stabilizer pH 1 20 mMHistidine-Chloride 150 mM NaCl 6.0 2 20 mM Histidine-Acetate

The stability study results of the two types of formulations containinghistidine-chloride or histidine-acetate using Mab1 and Mab2 are shown inFIGS. 1 to 3 . The results demonstrated that Mab1 was slightly morestable in histidine-chloride than in histidine-acetate during bothliquid storage and freeze-thawing. The result of liquid storage showedthat Mab2 was about twofold more stable in histidine-chloride than inhistidine-acetate.

Example 2 Assessment of the Stabilization Effect of Counter Ion SpeciesUsing Mab1 (1)

In general, hydrochloric acid has been used as a counter ion species tohistidine and arginine. According to a previous report(PCT/US2005/037471), results of assessing acetic acid, phosphoric acid,and sulfuric acid as a counter ion species to histidine showed thatacetic acid has a superior stabilizing effect when used as a counter ionspecies to histidine. However, as described in Example 1 above, thepresent invention demonstrated that hydrochloric acid was slightly moresuperior than acetic acid as a counter anion species to histidine whenused for Mab1 and Mab2. Hydrochloric acid is a common counter anionspecies, while hydrochloric acid has been reported to have a tendency ofcorroding stainless steel which is generally used for containers (Dent.Mater. 17:409-414 (2001); J. Pharm. Sci. 86:1250-1255 (1997)). Besides,it has been reported that the pH tends to alter in acetic acid becauseof its volatility (Injectable Drug Development, Authors: Pramod K. Gupta(Editor), Gayle A. Brazeau, Gayle A).

Thus, in this Example, the present inventors searched for involatilecounter ions that are not corrosive for stainless steel and have asuperior stabilization effect than acetic acid and hydrochloric acid, asa counter anion species in the buffer for Mab1 and Mab2. The presentinventors assessed anion species other than hydrochloric acid, aceticacid, phosphoric acid, and sulfuric acid previously reported inPCT/US2005/037471. Specifically, aspartic acid and glutamic acid, whichare both amino acids, were assessed as counter ion species. As describedin Example 1, histidine-chloride was demonstrated to have a moresuperior stabilization effect than histidine-acetate in both Mab1 andMab2, so the stabilization effect of counter ion species was compared tothat of hydrochloric acid. Specifically, according to the threeformulations shown in Table 2, the effect of hydrochloric acid, asparticacid, and glutamic acid on stability were assessed as a counter anionspecies to histidine as a buffer or arginine as a stabilizer using Mab1.

Each formulation was prepared by the same method as described inExample 1. Mab1 was dialyzed against the solution of each formulation(Table 2) overnight. Then, each solution was concentrated, and the finalconcentration of Mab1 was adjusted to 200 mg/ml. The freeze-thawingstudy was carried out by conducting ten cycles of slow freeze-thawing(freezing at −20° C. followed by thawing at room temperature). Themethod for preparing each formulated solution is described below.Formulation No. 3 sample was prepared as follows: L-histidine andL-arginine were dissolved in MilliQ water at 20 mM and 50 mM,respectively, and the solutions were titrated to pH 6 with 1 Nhydrochloric acid. Formulation Nos. 4 and 5 samples were prepared asfollows: L-histidine, L-arginine, and L-aspartic acid or L-glutamic acidwere dissolved in MilliQ water at 20 mM, 50 mM, and 60 mM, respectively,and then the solutions were titrated to pH 6 with a 30 to 40 mML-aspartic acid or L-glutamic acid solution. The amount of aggregate ineach sample after freeze-thawing or storage at −20° C., 25° C., and 5°C. was calculated by the area percentage method using size exclusionchromatography (SEC).

TABLE 2 Formulation list No. Buffer Stabilizer pH 3 20 mMHistidine-Chloride 50 mM Arg-Chloride 6.0 4 20 mM Histidine-Aspartate 50mM Arg-Aspartate 5 20 mM Histidine-Glutamate 50 mM Arg-Glutamate

The increased amount (%) of aggregate during freeze-thawing or storageat −20° C., 25° C., and 5° C. for each formulation is shown in FIGS. 4to 7 . The increased amount (%) of aggregate during storage at 5° C. and25° C., showed that stability was enhanced in the order of: glutamicacid=aspartic acid>hydrochloric acid as counter ion species to histidineand arginine. Thus, it was demonstrated that the stability of Mab1 wasimproved by using aspartic acid or glutamic acid as a counter ionspecies, instead of hydrochloric acid. The same tendency was seen infreeze-thawing and frozen storage. The increased amount (%) of aggregateduring storage at −20° C. for three months with the glutamic acidformulation, aspartic acid formulation, or hydrochloric acid formulationwas about 0.8%, 1.2%, or 3.0%, respectively. Thus, the stabilizationeffect of glutamic acid was slightly stronger than that of asparticacid.

Examples 1 and 2 demonstrated that when used as a counter anion species,glutamic acid and aspartic acid have a superior Mab1-stabilizing effectthan hydrochloric acid and acetic acid. There is no report demonstratingthat glutamic acid and aspartic acid are volatile or corrosive forstainless steel. Thus, glutamic acid and aspartic acid are found to bepromising as a counter anion species for Mab1. Specifically,histidine-glutamate and histidine-aspartate are superior as a bufferthan histidine-chloride and histidine-acetate, and arginine-glutamateand arginine-aspartate are more superior as a stabilizer thanarginine-chloride and arginine-acetate. [Example 3] Assessment of theStabilization Effect of Counter Ion Species Using Mab2 (1)

As described in Example 1, Mab2 was found to be more stable inhistidine-chloride buffer than histidine-acetate buffer (like Mab1, FIG.2 ). Furthermore, as described in Example 2, the stability in liquid andfrozen conditions of Mab1 was significantly improved when aspartic acidor glutamic acid was used instead of hydrochloric acid as a counter ionspecies to histidine and arginine. In particular, the stability infrozen conditions of Mab1 was greatly improved (to about threefold) whenglutamic acid was used instead of hydrochloric acid as a counter ionspecies to histidine and arginine. In this context, glutamic acid andhydrochloric acid were assessed as a counter ion species to histidinefor their ability to stabilize Mab2 during freeze-thawing.Arginine-containing formulations which have high stabilization effectwere also assessed at the same time, and used as a control to comparethe stabilization effect observed when glutamic acid was used as acounter ion species to histidine.

Each formulation was prepared by the same method as described inExample 1. Mab2 was dialyzed overnight against each formulated solution(Table 3). Then, each solution was concentrated, and the finalconcentration of Mab2 was adjusted to about 40 to 230 mg/ml. The methodfor preparing the each formulated solution is described below.Formulation Nos. 6 and 8 samples were prepared as follows: L-histidineand L-arginine (formulation No. 8 alone) were each dissolved in MilliQwater at 50 mM, and the solutions were titrated to pH 6 with 1 Nhydrochloric acid. Formulation No. 7 sample was prepared as follows:L-histidine and L-glutamic acid were dissolved in MilliQ water at 50 mMand 25 mM, respectively, and then the solution was titrated to pH 6 witha 30 to 40 mM L-glutamic acid solution. The concentration of Mab2 ineach formulation after sample preparation is shown in Table 4. Thefreeze-thawing study was carried out with ten cycles of freezing at −20°C. followed by thawing at room temperature (slow freeze-thawing).Following slow freeze-thawing, the amount of aggregate in eachformulation was calculated by the area percentage method using sizeexclusion chromatography (SEC).

TABLE 3 Formulation list No. Buffer Stabilizer pH 6 50 mMHistidine-Chloride — 6.0 7 50 mM Histidine-Glutamate 8 50 mMHistidine-Chloride 50 mM Arginine-Chloride

TABLE 4 Measurement result: List of Mab2 concentration in solution ofeach formulation Mab2 Concentration No. Buffer Stabilizer pH (mg/mL) 6A50 mM Histidine-Chloride — 6.0 47 6B 70 6C 97 6D 122 6E 143 6F 164 6G186 6H 229 7A 50 mM Histidine-Glutamate 46 7B 72 7C 97 7D 119 7E 144 7F165 7G 196 8A 50 mM Histidine-Chloride 50 mM 44 8B Arginine- 68 8CChloride 94 8D 120 8E 144 8F 168 8G 192

The increased amount (%) of aggregate for each formulation in thefreeze-thawing study is shown in FIG. 8 . The results demonstrated thatby using glutamic acid instead of hydrochloric acid as a counter ionspecies to histidine, the stability of Mab2 was improved to about twotimes. In addition, the stabilization effect of glutamic acid wascomparable to that of 50 mM arginine chloride, which is a conventionalstabilizer. Thus, glutamic acid alone was demonstrated to exert highstabilization effect as a counter ion species.

Meanwhile, it is believed that the osmotic pressure ratio of the bufferis preferably close to isotonic 1.0 in formulations for subcutaneousadministration because of the injection pain. The stability duringfreeze-thawing was comparable between 50 mM histidine-chloride/50 mMarginine-chloride and 50 mM histidine-glutamate. The osmotic pressure ofthe buffer in the latter was about 100 mOsm lower than in the former.Thus, as described above, when the stability is improved by usingaspartic acid or glutamic acid as a counter ion species, stability canbe enhanced alone without increasing the osmotic pressure. This can be agreat advantage in developing formulations for subcutaneousadministration.

Meanwhile, arginine, sugars, and such are added to improve the stabilityof formulations during storage. However, when the osmotic pressure isgreater than the isotonic level, it may cause injection pain atsubcutaneous administration. Thus, such stabilizers must be added byconsidering the osmotic pressure (Injectable Drug Development, Authors:Pramod K. Gupta (Editor), Gayle A. Brazeau, Gayle A; Challenges in thedevelopment of high protein concentration formulations, J Pharm Sci,2004, 93(6), 1390-1402). When hydrochloric acid or acetic acid is addedas a counter anion species to histidine or arginine, neitherhydrochloric acid nor acetic acid has the effect of stabilizing Mab1.Thus, hydrochloric acid and acetic acid only produce the effect ofincreasing the osmotic pressure. Accordingly, from the viewpoint ofosmotic pressure, the concentration of ion species that do not have thestabilization effect should be minimized in the formulations.Specifically, also from the viewpoint of osmotic pressure, absence ofhydrochloric acid and acetic acid is preferred. Histidine-glutamate andhistidine-aspartate are superior as a buffer than histidine-chloride andhistidine-acetate; and arginine-glutamate and arginine-aspartate aresuperior as a stabilizer than arginine-chloride and arginine-acetate.

Example 4 Assessment of the Stabilization Effect of Counter Ion SpeciesUsing Mab1 (2)

As described in Examples 2 and 3, by using glutamic acid as a counterion species to histidine and arginine, the stability of Mab1 wassignificantly improved to about two to three folds, in particular duringfrozen storage. In this context, a storage stability study at −20° C.was conducted to assess the stability of Mab1 at −20° C. storage whenglutamic acid was used as a counter ion species to histidine andarginine, and a sugar (trehalose) as a stabilizer. Liquid storage andfreeze-thawing study were also carried out at the same time.

Each formulation was prepared as follows: Mab1 was dialyzed overnightagainst each formulated solution (Table 5); then, the solutions wereconcentrated, and the final concentration of Mab1 was adjusted to 200mg/ml. The method for preparing each formulated solution is describedbelow. L-Histidine, L-arginine, L-glutamic acid, and trehalose weredissolved in MilliQ water at 100 mM, 50 mM, 100 mM, and 0 to 150 mM,respectively, and then the solutions were titrated to pH 6 with 30 to 40mM L-glutamic acid solution. The freeze-thawing study was carried outwith ten cycles of freezing at −20° C. followed by thawing at roomtemperature (slow freeze-thawing). The amount of aggregate in eachformulation after freeze-thawing or storage at −20° C. and 25° C. wascalculated by the area percentage method using size exclusionchromatography (SEC).

TABLE 5 Formulation list No. Buffer Stabilizer Sugar pH  9 100 mM 50 mM— 6.0 10 Histidine- Arginine-Glutamate  50 mM Trehalose 11 Glutamate 100mM Trehalose 12 150 mM Trehalose

The increased amount (%) of aggregate of each formulation after −20° C.storage, freeze-thawing, and 25° C. storage is shown in FIGS. 9 to 11 .By adding 50 mM or more trehalose, a formulation with which theaggregate is hardly increased during storage at −20° C. andfreeze-thawing was obtained, as seen from FIGS. 9 to 11 . Furthermore,the trehalose concentration-dependent stabilization effect was observedin the liquid storage at 25° C. As described above, the presentinventors discovered simple formulations consisting only of amino acidsand sugar, which contribute to the stabilization during both liquidstorage and frozen storage.

Example 5 Assessment of the Stabilization Effect of Counter Ion SpeciesUsing Mab2 (2)

As described in Example 1, Mab2 was shown to be more stabilized byhistidine-chloride than by histidine-acetate, like Mab1 (FIG. 2 ).Furthermore, as described in Example 2, the stability in liquid and infrozen conditions of Mab1 was significantly improved when aspartic acidor glutamic acid was used instead of hydrochloric acid as a counter ionspecies to histidine and arginine. In this context, the stability ofMab2 during liquid storage (25° C.) was used to assess hydrochloric acidand glutamic acid as a counter ion species to histidine.Arginine-containing formulations which have high stabilization effectwere also assessed at the same time, and used as a control to comparethe stabilization effect observed when glutamic acid is used as acounter ion species to histidine.

Each formulation was prepared by the same method as described inExample 1. Mab2 was dialyzed overnight against each formulated solution(Table 3). Then, the solutions were concentrated, and the finalconcentration of Mab2 was adjusted to about 40 to 230 mg/ml. The methodfor preparing the formulated solutions was the same as described inExample 3. The concentration of Mab2 in each formulation after samplepreparation is shown in Table 4. The amount of aggregate in eachformulation during two to four weeks of storage at 25° C. was calculatedby the area percentage method using size exclusion chromatography (SEC).

The increased amount (%) of aggregate for each formulation after storageat 25° C. is shown in FIG. 12 . The results showed that, unlike in thefreeze-thawing study, the stability of Mab2 was not altered even whenglutamic acid was used instead of hydrochloric acid as a counter ionspecies to histidine. The pIs of Mab1 and Mab2 are 5.8 and 9.3,respectively. This suggests that the stabilization effect of counter ionspecies on antibodies with low pI in liquid storage was significant.

Example 6 Assessment of Stabilization Effect of Counter Ion SpeciesUsing Mab1, Mab2, Mab3, Mab4, and Mab5

Mab3 is a bispecific antibody of factor IX and factor X and has anIgG4-derived constant region. Furthermore, its pI value has been loweredto 6.8 by altering the amino acid sequence.

Mab4 is a humanized anti-NR10 antibody (completely humanized NS22antibody prepared according to the method described in Example 12 of WO2009/072604), and its antibody class is IgG2. Its pI value has beenlowered to 5.6 by altering the amino acid sequence.

Mab5 is a humanized anti-glypican 3 antibody (it was humanized by themethod described in Example 24 of WO 2006/006693 and its L chain wasaltered by the method of Example 25). Its antibody class is IgG1.

As described in Examples 2 and 3, the stability in liquid and frozenconditions of Mab1 and Mab2 were demonstrated to be significantlyimproved when aspartic acid or glutamic acid was used instead ofhydrochloric acid as a counter ion species to histidine and arginine.Then, Mab1 and Mab2 as well as Mab3, Mab4, and Mab5 which are antibodiesmodified to have an isoelectric point of 5 to 8 were used to assess thesolution stability and freeze-thawing stability when hydrochloric acidand aspartic acid is used as a counter ion species to histidine andarginine. The pIs of Mab1, Mab2, Mab3, Mab4, and Mab5 are shown in Table6 below.

TABLE 6 Sample Mab1 Mab2 Mab3 Mab4 Mab5 pI 5.8 9.4 6.8 5.6 9.0

Samples were prepared as follows: Mab1, Mab2, Mab3, Mab4, and Mab5 weredialyzed against each dialysis buffer (Table 7) overnight. Then, eachantibody solution was concentrated, and a stock buffer for eachformulation (Table 8) was added thereto so that the final antibodyconcentration was adjusted to about 100 to 190 mg/ml. A list of theformulated solutions prepared as described above is shown in Table 9.For each formulation, liquid storage study at 25° C. and freeze-thawingstudy were carried out. The freeze-thawing study was carried out in tencycles of freezing at −20° C. followed by thawing at 25° C. (slowfreeze-thawing). Following slow freeze-thawing, the amount of aggregatein each sample was calculated by the area percentage method using sizeexclusion chromatography (SEC).

TABLE 7 No. Sample Dialysis buffer pH 13 Mab3 Water 6.0 14 Mab3 15 Mab450 mM Histidine-Chloride 16 Mab4 50 mM Histidine-Aspartate 17 Mab2 20 mMHistidine-Chloride 18 Mab2 19 Mab1 20 Mab1 21 Mab3 Water 22 Mab3 23 Mab520 mM Histidine-Chloride 24 Mab5

TABLE 8 No. Sample Stock buffer pH 13 Mab3 500 mM Histidine-Chloride 6.014 Mab3 500 mM Histidine-Aspartate 15 Mab4 50 mM Histidine-Chloride 16Mab4 50 mM Histidine-Aspartate 17 Mab2 20 mM Histidine-Chloride, 500 mMArginine-Chloride 18 Mab2 20 mM Histidine-Chloride, 500 mMArginine-Aspartate 19 Mab1 20 mM Histidine-Chloride, 500 mMArginine-Chloride 20 Mab1 20 mM Histidine-Chloride, 500 mMArginine-Aspartate 21 Mab3 200 mM Histidine-Chloride, 500 mMArginine-Chloride 22 Mab3 200 mM Histidine-Chloride, 500 mMArginine-Aspartate 23 Mab5 20 mM Histidine-Chloride, 500 mMArginine-Chloride 24 Mab5 20 mM Histidine-Chloride, 500 mMArginine-Aspartate

TABLE 9 Mab concentration No. Sample formulation pH (mg/mL) 13 Mab3 50mM Histidine-Chloride 6.0 100 14 Mab3 50 mM Histidine-Aspartate 15 Mab450 mM Histidine-Chloride 16 Mab4 50 mM Histidine-Aspartate 17 Mab2 20 mMHistidine-Chloride, 190 50 mM Arginine-Chloride 18 Mab2 20 mMHistidine-Chloride, 50 mM Arginine-Aspartate 19 Mab1 20 mMHistidine-Chloride, 110 50 mM Arginine-Chloride 20 Mab1 20 mMHistidine-Chloride, 50 mM Arginine-Aspartate 21 Mab3 20 mMHistidine-Chloride, 50 mM Arginine-Chloride 22 Mab3 20 mMHistidine-Chloride, 50 mM Arginine-Aspartate 23 Mab5 20 mMHistidine-Chloride, 120 50 mM Arginine-Chloride 24 Mab5 20 mMHistidine-Chloride, 50 mM Arginine-Aspartate

The result on the increased amount (%) of aggregate in each formulationafter freeze-thawing or liquid storage at 25° C. is shown in FIGS. 13 to20 . Comparison of the increased amounts of aggregate during liquidstorage at 25° C. demonstrated that the stability was comparable betweenthe histidine-aspartate formulation and histidine-chloride formulation,and between the arginine-aspartate formulation and arginine-chlorideformulation (FIGS. 13, 15, 17 , and 19).

Meanwhile, comparison of the increased amounts of aggregate afterfreeze-thawing revealed that the stability with the histidine-aspartateformulation was two or more times higher than that with thehistidine-chloride formulation, and the stability with thearginine-aspartate formulation was higher than that with thearginine-chloride formulation (FIGS. 14, 16, 18 , and 20). Thus, thestability in frozen condition of antibody was demonstrated to besignificantly improved by using aspartic acid instead of hydrochloricacid as a counter ion species to histidine or arginine.

Example 7 Assessment of Stabilization Effect of Counter Ion SpeciesUsing Mab1, Mab2, Mab3, Mab4, and Mab5

As described in Examples 2, 3, and 6, the stability in liquid and frozenconditions of antibody was demonstrated to be significantly improvedwhen aspartic acid or glutamic acid was used instead of hydrochloricacid as a counter ion species in the histidine formulation. Then,hydrochloric acid and aspartic acid were used as a counter ion speciesin the tris(hydroxymethyl) aminomethane (Tris) formulation, and assessedfor the liquid storage stability and freeze-thawing stability usingMab1, Mab2, Mab3, Mab4, and Mab5.

Samples were prepared as follows: Mab1, Mab2, Mab3, Mab4, and Mab5 weredialyzed against each dialysis buffer (Table 10) overnight. Then, eachantibody solution was concentrated, and a stock buffer for eachformulation (Table 11) was added thereto so that the final antibodyconcentration was adjusted to about 100 to 110 mg/ml. A list of theformulated solutions prepared as described above is shown in Table 12.For each formulated solution, storage study at 25° C. and freeze-thawingstudy were carried out. The freeze-thawing study was carried out in tencycles of freezing at −20° C. followed by thawing at 25° C. (slowfreeze-thawing). Following slow freeze-thawing, the amount of aggregatein each sample was calculated by the area percentage method using sizeexclusion chromatography (SEC).

TABLE 10 No. Sample Dialysis buffer pH 25 Mab1 20 mM Tris-Chloride 6.526 Mab1 20 mM Tris-Aspartate 27 Mab2 20 mM Tris-Chloride 28 Mab2 20 mMTris-Aspartate 29 Mab3 Water 30 Mab3 Water 31 Mab4 20 mM Tris-Chloride32 Mab4 20 mM Tris-Aspartate 33 Mab5 20 mM Tris-Chloride 34 Mab5 20 mMTris-Aspartate

TABLE 11 No. Sample Stock buffer pH 25 Mab1 20 mM Tris-Chloride, 500 mMArginine-Chloride 6.5 26 Mab1 20 mM Tris-Chloride, 500 mMArginine-Aspartate 27 Mab2 20 mM Tris-Chloride, 500 mM Arginine-Chloride28 Mab2 20 mM Tris-Chloride, 500 mM Arginine-Aspartate 29 Mab3 200 mMTris-Chloride, 500 mM Arginine-Chloride 30 Mab3 200 mM Tris-Chloride,500 mM Arginine-Aspartate 31 Mab4 20 mM Tris-Chloride, 500 mMArginine-Chloride 32 Mab4 20 mM Tris-Chloride, 500 mM Arginine-Aspartate33 Mab5 20 mM Tris-Chloride, 500 mM Arginine-Chloride 34 Mab5 20 mMTris-Chloride, 500 mM Arginine- Aspartate

TABLE 12 Mab concentration No. Sample formulation pH (mg/mL) 25 Mab1 20mM Tris-Chloride, 6.5 100 50 mM Arginine-Chloride 26 Mab1 20 mMTris-Chloride, 50 mM Arginine-Aspartate 27 Mab2 20 mM Tris-Chloride, 50mM Arginine-Chloride 28 Mab2 20 mM Tris-Chloride, 50 mMArginine-Aspartate 29 Mab3 20 mM Tris-Chloride, 50 mM Arginine-Chloride30 Mab3 20 mM Tris-Chloride, 50 mM Arginine-Aspartate 31 Mab4 20 mMTris-Chloride, 110 50 mM Arginine-Chloride 32 Mab4 20 mM Tris-Chloride,50 mM Arginine-Aspartate 33 Mab5 20 mM Tris-Chloride, 50 mMArginine-Chloride 34 Mab5 20 mM Tris-Chloride, 50 mM Arginine-Aspartate

The result on the increased amount (%) of aggregate in each formulationafter freeze-thawing or liquid storage at 25° C. is shown in FIGS. 21and 22 . Comparison of the increased amounts of aggregate during liquidstorage at 25° C. demonstrated that the stability was comparable betweenthe Tris-aspartate/arginine-aspartate formulation andTris-chloride/arginine-chloride (FIG. 21 ).

Meanwhile, comparison of the increased amounts of aggregate afterfreeze-thawing revealed that the stability with theTris-aspartate/arginine-aspartate formulation was higher than that withthe Tris-chloride/arginine-chloride formulation (FIG. 22 ). Thus, thestability in frozen condition of antibody was also demonstrated to besignificantly improved by using aspartic acid instead of hydrochloricacid as a counter ion species in the Tris formulation.

Example 8 Assessment of Stabilization Effect of Counter Ion Species toTris Using Mab1, Mab2, and Mab3

As described in Example 7, the stability in frozen condition of antibodywas demonstrated to be significantly improved when aspartic acid wasused instead of hydrochloric acid as a counter ion species in the Trisformulation. Then, hydrochloric acid and aspartic acid were used as acounter ion species to Tris, and assessed for the liquid storagestability and freeze-thawing stability using Mab1, Mab2, and Mab3.

Samples were prepared as follows: Mab1, Mab2, and Mab3 were dialyzedagainst each dialysis buffer (Table 13) overnight. Then, each antibodysolution was concentrated to 100 mg/ml or a higher concentration, andeach dialysate was added thereto so that the final antibodyconcentration was adjusted to about 100 mg/ml. A list of the formulatedsolutions prepared as described above is shown in Table 14. Storagestudy at 25° C. and freeze-thawing study were carried out using eachformulated solution. The freeze-thawing study was carried out in tencycles of freezing at −20° C. followed by thawing at 25° C. (slowfreeze-thawing). Following slow freeze-thawing, the amount of aggregatein each sample was calculated by the area percentage method using sizeexclusion chromatography (SEC).

TABLE 13 No. Sample Dialysis buffer pH 35 Mab1 50 mM Tris-Chloride 6.536 Mab1 50 mM Tris-Aspartate 37 Mab2 50 mM Tris-Chloride 38 Mab2 50 mMTris-Aspartate 39 Mab3 50 mM Tris-Chloride 40 Mab3 50 mM Tris-Aspartate

TABLE 14 Mab concentration No. Sample Formulation pH (mg/mL) 35 Mab1 50mM Tris-Chloride 6.5 100 36 Mab1 50 mM Tris-Aspartate 37 Mab2 50 mMTris-Chloride 38 Mab2 50 mM Tris-Aspartate 39 Mab3 50 mM Tris-Chloride40 Mab3 50 mM Tris-Aspartate

The result on the increased amount (%) of aggregate after freeze-thawingor liquid storage at 25° C. in each formulation is shown in FIGS. 23 and24 . Comparison of the increased amounts of aggregate based on thisresult demonstrated that during both liquid storage at 25° C. and duringfreeze-thawing, the stability with the Tris-aspartate formulation washigher than that with the Tris-chloride formulation, and the stabilityduring freeze-thawing was in particular two or more times higher. Thus,the antibody stability was also demonstrated to be significantlyimproved by using aspartic acid instead of hydrochloric acid as acounter ion species to Tris used as a buffering agent.

1.-23. (canceled)
 24. A method for suppressing aggregate formationduring liquid or frozen storage storage of an antibody-comprisingformulation by using aspartic acid or glutamic acid as a counter ionspecies to a tris(hydroxymethyl) aminomethane buffer in the formulation,wherein the pH of the formulation is 5.5 to 8 and wherein theconcentration of the antibody in the formulation is 50 mg/mL or more.25. (canceled)
 26. A method for suppressing aggregate formation duringliquid or frozen storage of an antibody-comprising formulation by usingaspartic acid or glutamic acid as a counter ion species to a stabilizerin the formulation, wherein the concentration of the antibody in theformulation is 50 mg/mL or more.
 27. (canceled)
 28. The method of claim24, wherein the method suppresses aggregate formation during liquidstorage.
 29. The method of claim 24, wherein the method suppressesaggregate formation during frozen storage.
 30. The method of claim 24,wherein the antibody has been modified to have an isoelectric point (pI)of between 5.0 to 8.0.
 31. The method of claim 24, wherein theconcentration of the antibody in the formulation is 50 to 250 mg/ml. 32.The method of claim 24, wherein formulation contains 5 mM or less, 2 mMor less, or 1 mM or less of chloride and acetate ions.
 33. The method ofclaim 24, wherein the formulation further comprises a sugar.
 34. Themethod of claim 24, wherein the tris(hydroxymethyl) aminomethane bufferconcentration is 5 to 100 mM.
 35. The method of claim 28, wherein theviscosity of the liquid formulation is 30 mPa s or less.
 36. The methodof claim 28, wherein the liquid formulation is stable at 2° C. to 8° C.for at least six months.
 37. The method of claim 29, wherein theformulation is frozen stored at −30° C. to −10° C.
 38. The method ofclaim 24, wherein the formulation has not been subjected tolyophilization.
 39. The method of claim 26, wherein the methodsuppresses aggregate formation during liquid storage.
 40. The method ofclaim 26, wherein the method suppresses aggregate formation duringfrozen storage.
 41. The method of claim 26, wherein the antibody hasbeen modified to have an isoelectric point (pI) of between 5.0 to 8.0.42. The method of claim 26, wherein the concentration of the antibody inthe formulation is 50 to 250 mg/ml.
 43. The method of claim 26, whereinformulation contains 5 mM or less, 2 mM or less, or 1 mM or less ofchloride and acetate ions.
 44. The method of claim 26, wherein theformulation further comprises a sugar.
 45. The method of claim 39,wherein the viscosity of the liquid formulation is 30 mPa s or less. 46.The method of claim 39, wherein the liquid formulation is stable at 2°C. to 8° C. for at least six months.
 47. The method of claim 40, whereinthe formulation is frozen stored at −30° C. to −10° C.
 48. The method ofclaim 26, wherein the formulation has not been subjected tolyophilization.
 49. The method of claim 26, wherein the counter ionspecies is aspartic acid.
 50. The method of claim 26, wherein thecounter ion species is glutamic acid.
 51. The method of claim 26,wherein the counter ion species is aspartic acid and glutamic acid. 52.The method of claim 26, wherein the stabilizer is selected fromargininamide, lysine, meglumine, spermine, spermidine, magnesium,calcium, sodium, potassium, and arginine.
 53. The method of claim 26,wherein the concentration of the stabilizer is 5 to 300 mM or 25 to 200mM.
 54. The method of claim 26, wherein the stabilizer is arginine. 55.The method of claim 54, wherein the arginine concentration is 25 to 200mM or 50 to 150 mM.